Ship 1 Modulators

ABSTRACT

The present invention includes the use of pelorol, related compounds and pharmaceutical compositions thereof as modulators of SHIP 1 activity. This invention also provides novel terpene compounds capable of modulating SHIP 1 activity and methods of synthesis thereof.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to SHIP 1, a negative regulator of cellproliferation and survival and immune cell activation.

BACKGROUND OF THE INVENTION

SH₂-containing inositol 5-phosphatase (SHIP 1), selectively hydrolyzesthe 5-phosphate from inositol 1,3,4,5-tetraphosphate (IP4) andphosphatalidylinositol 3,4,5-triphosphate (PIP3). U.S. Pat. No.6,238,903 discloses that SHIP 1 is an enzyme regulator of signalingpathways that control gene expression, cell proliferation,differentiation, activation, and metabolism, particularly of the Ras andphospholipid signaling pathways. SHIP 1 plays an important role incytokine and immune receptor signal tansduction. SHIP 1 disrupted (SHIP1−/−) mice exhibit a myeloproliferative phenotype characterized byoverproduction of granulocytes and macrophages¹. SHIP 1−/− mast cellsare more prone to IgE and Steel factor induced degranulation, while SHIP1−/− B cells are resistant to negative regulation by Fc RIIB. SHIP 1 isalso involved in the pathogenesis of chronic myelogenous leukemia².

Compounds that specifically modulate the activity of SHIP 1 would beuseful in the treatment of cell proliferation, hematopoietic and immunedisorders, as well as for manipulating SHIP 1 mediated pathways duringinvestigatory and drug discovery testing. To date, no structure of asmall molecule SHIP 1 specific modulator has been disclosed.

A sesquiterpene compound termed pelorol may be obtained from variousmarine sponge species, including Petrosaspongia metachromia andDactylospongia elegans. Kwak et al. and Goclik et al. each disclosed thestructure of pelorol and its extraction from different marinesponges.^(4,5) Pelorol was reported as having weak antitrypanosomal andantiplasmodial effects⁵. The precise structure of pelorol is as follows,with Me representing a methyl group and relative configuration of chiralatoms (C-5, 8, 9 and 10) shown.

Some reduced and substituted chrysene derivatives similar to pelorol andhaving the characteristic gem substituted non-aromatic ring of pelorolare known as intermediates or derivatives in the preparation of variouspolycyclic polyprenols found in shale⁶⁻¹², in the preparation oftaxodione¹³, and in the compound1,2,3,4,4a,4b,5,6,10b,11,12,12a-dodecahydro-1,1-dimethyl-chrysene¹⁴.None of these chrysene derivatives are known to have biologicalactivity.

SUMMARY OF THE INVENTION

This invention is based on the discovery that pelorol and relatedcompounds are capable of modulation of SHIP 1 activity.

Some embodiments of this invention provide novel compounds of Formula Iand salts thereof. Compounds of Formula I have the structure:

wherein;

R₁ and R₂ are independently selected from the group consisting of: —CH₃,—CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′;

R₃ and R₄ are independently selected from the group consisting of: H,—CH₃, —CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′;

Q is selected from the group consisting of: —CH₂—, —CY₁Y₂—, —CH₂CH₂—,—CH═CH—, —C₁Y₂CY₃Y₄—, —CH₂CH₂CH₂—, —CH═CHCH₂—, —CH═CHCY₁Y₂—, and—C₁Y₂CY₃Y₄CY₅Y₆—; where Y₁, Y₂, Y₃, Y₄, Y₅, and Y₆ are independentlyselected from the group consisting of: H, F, Br, Cl, I, OH, OR′, and SH;or any one group of Y₁/Y₂, Y₃/Y₄, and Y₅/Y₆ may be ═O; or Y₁/Y₃ may forman epoxide; and, at least one of Y₁, Y₂, Y₃, Y₄, Y₅ and Y₆ when present,is not H;

X₁, X₂, X₃, and X₄ are independently selected from the group consistingof: H, R, OH, —OR, —CO₂H, —CO₂R′, F, Br, Cl, I, —CN, —SO₃H, —OSO₃H, NO₂,NH₂, —NHR, and —NR₂; where R is a linear, branched, or cyclic, saturatedor unsaturated one to ten carbon alkyl group that is unsubstituted or issubstituted with one or more of: OH, ═O, SH, F, Br, Cl, I, NH₂, —NHR′,—NR′₂, NO₂, —CO₂H, —CO₂R′, and epoxide;

and R′ is a linear, branched, or cyclic, saturated or unsaturated one toten carbon alkyl group that is unsubstituted or substituted with one ormore of: OH, ═O, SH, F, Br, Cl, I, NH₂, —NHR″, —NR″₂, NO₂ and —CO₂Hwhere R″ is a linear, branched, or cyclic, saturated or unsaturated oneto ten carbon alkyl group.

Novel compounds of Formula I of this invention do not include theprecise structures of previously described gem substituted chrysenederivatives. These previously described compounds include pelorol andcompounds having the following structures in which Me is methyl:

Alternately defined, this invention excludes such previously knownspecific compounds of Formula I in which each of R₁-R₄ are methyl; Q is—CH₂CH₂—; and, X₁-X₄ is according to any one of the followingdefinitions:

(a) X₁ and X₂=OH, X₃=H, and X₄=—COOCH₃;

(b) X₁, X₂, X₃ and X₄=H;

(c) X₁, X₂, and X₄=H, and X₃=CH₃;

(d) X₁, X₃, and X₄=H, and X₂=CH₃;

(e) X₂, X₃, and X₄=H, and X₁=CH₃; and

(f) X₁ and X₄=H, X₂ and X₃=OCH₃.

Also excluded is a compound of Formula I in which R₁ and R₂=CH₃; R₃ andR₄=H; Q=—CH₂CH₂—; and each of X₁-X₄ is H.

Some embodiments of this invention provide a pharmaceutical compositioncomprising one or more compounds of Formula I or pharmaceuticallyacceptable salts thereof, and a pharmaceutically acceptable carrier.Such compositions may comprise previously known compounds of Formula Iwhich have not been known as biologically active compounds suitable forpharmaceutical use.

Some embodiments of this invention provide a method of treatment orprevention of an immune, inflammatory, or neoplastic disorder orcondition, comprising administering to a patient in need of suchtreatment or prevention, an effective amount of a compound of Formula Ior pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition of this invention.

Some embodiments of this invention provide the use of a compound ofFormula I or pharmaceutically acceptable salt thereof for modulation ofSHIP 1 activity and for preparation of agents for the modulation of SHIP1 activity. Such modulation may be in vitro or in vivo. Agents for invivo use include a pharmaceutical composition of this invention as wellas agents adapted for in vitro use. The modulation may be for atreatment or prevention of an immune, inflammatory, or neoplasticcondition or disorders as described above.

Some compounds of Formula I may be prepared in whole or in part byfractionating biological extracts or by derivatizing availablecompounds. Alternately, compounds of Formula I may be prepared by totalsynthesis.

Some embodiments of this invention provide a method of making a compoundof Formula IA

in which R₁-R₄, X₁, X₃, and X₄ are as defined for Formula I, L′ is aC₁-C₄ saturated or unsaturated alkyl linking group; and A is anactivating group; comprising reacting a compound of Formula IIA or IIB:

in which L is absent or is a C₁-C₃ saturated or unsaturated alkyllinking group and E and E′ are electrophilic reactive groups; with acompound of Formula III

in which Nu is a group that renders the compound of Formula IIInucleophilic at Nu, followed by optional reduction and by hydrolysis, toproduce a compound of Formula IV

and condensing the compound of Formula IV to produce a compound ofFormula IA.

L′ in compounds of Formula IA may optionally be changed or derivatizedto form a desired component Q of Formula I. For example, component L′ incompounds of Formula IA produced by the preceding method may havedifferent degrees of saturation or different substituents as compared toQ in a compound of Formula I. In order to reduce the number of atoms inthe ring, a compound having an unsaturated L′ group could be subjectedto oxidizing and reduction steps to reduce the size of the ring inFormula I comprising Q. In addition, functionalities such as ketone,hydroxyl, or other groups may be added to L′ to form a desired Qcomponent.

Preferred electrophilic reactive groups for E are lactone, ester, andthioester. A preferred group for E′ is carboxyl. More preferably,compounds of Formulas IIA and IIB are as follows.

Even more preferably, compounds of Formulas IIA and IIB are as follows:

A preferred Nu in compounds of Formula III is lithium which may besubstituted onto the ring for a halogen such as bromine. Preferably, Ain the compound of Formula III is an activating group such as —OMe orNHAc (Me=methyl and Ac=acetyl) which group may be subsequently convertedto a desired substituent for X₂ in compounds of Formula I. Substitutentsmay also be protected, where appropriate with a protecting group such asTBS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the effect of sponge extracts on SHIP 1enzyme activity in vitro.

FIG. 2 is a graph depicting the effect of pelorol on macrophage nitricoxide (NO) production.

FIG. 3 is a graph depicting the effect of pelorol on IgE mediated mastcell activation.

DETAILED DESCRIPTION OF THE INVENTION

In this specification, the following abbreviations will appear: THF(tetrahydrofuran); n-buLi (n-butyl)-lithium); t-buLi(tert-butyl)-lithium); Ph₃PMe (methyl triphenyl posphonium bromide); PCC(pyridinium chlorochromate); Ac (acetyl); Me (methyl); Et (ethyl); prop.(propyl); but. (butyl); RT or, r.t. (room temperature); hr. (hour(s));DMSO (dimethylsulfoxide); DNFB (2,4-dinitrofluorobenzene); LPS(lipopolysaccarhide); TNF-α (Tumor Necrosis Factor Alpha); TBS(tert-butyl dimethylsilyl); and EA (ethyl acetate).

SHIP 1 Modulating Compounds

Compounds of Formula I have chiral centres at C-5, C-8, C-9 and C-10 andmay be chiral at C-4 depending upon whether R₁ and R₂ are different.Compounds of this invention include all stereoisomers and enantiomers ofcompounds of Formula I. Some embodiments have the same relativeconfiguration of chiral centres as does pelorol or are enantiomersthereof, namely: S, R, R, S; or R, S, S, R (at C-5, 8, 9 and 10respectively). Some embodiments have the same absolute configuration aspelorol at chiral centres. Some embodiments have the same relativeconfiguration as pelorol at C-5 and C-10 with independently variableconfigurations at C-8 and C-9. Some embodiments have the same relativeconfiguration as pelorol at C-5, C-8, and C-10 with variableconfiguration at C-9. In all cases, the configuration at C-4 (if chiral)may be variable or may be the same relative configuration to theremaining chiral centres as is shown in examples of structures ofcompounds of Formula I illustrated herein.

In various embodiments of this invention, the compounds may have thelimitations in Formula I described above or may have more specificlimitations with respect to substituents Q, R₁-R₄, and X₁-X₄. Anycombination of the following limitations is encompassed by thisinvention.

(a) Q may be as defined for Formula I except that Y₁₋₆ is limited to Hor halogen;

(b) Q may be limited to —CH₂—, —CH₂CH₂—, —CH═CH, —CH₂—CH₂CH₂— and—CH═CHCH₂—;

(c) Q may be limited to H or saturated moieties in the limitation ofFormula I, or according to the limitations of paragraph (a) or (b)above;

(d) Q may be limited to a one or two carbon skeleton within thelimitations of Formula I, or according to the limitations of any ofparagraphs (a)-(c) above;

(e) one or both of R₁ and R₂ may be limited to methyl, ethyl, —CH₂OH or—CH₂OR′; (f) R′ in one or both of R₁ and R₂ according to Formula I, orthe limitation of paragraph (e) above, may be limited to methyl, ethyl,propyl or butyl;

(g) one or both of R₁ and R₂ may be limited to methyl or ethyl;

(h) one or both of R₁ and R₂ may be limited to methyl;

(i) R and R′ in any one or more of X₁-X₄ may be limited to unsubstitutedmethyl, ethyl, propyl or butyl;

(j) one or more of X₁-X₃ may be limited to H, R, OH, OR, halogen,—CONH₂, —CONHR′, —COR′₂, NHR or NR₂ where R and R′ are limited as inFormula I, or R and R′ may be according to paragraph (i) above;

(k) one or more of X₁-X₃ is limited to H, OH, OR, —CONH₂, —CONHR′, and—COR′₂, where R and R′ are as in Formula I, or R and R′ may be limitedaccording to paragraph (i) above;

(l) one or more of X₁-X₃ may be limited to H, OH, and OCH₃;

(m) X₄ may be limited to H, R, OH, OR, CO₂H or —CO₂R′, with R and R′ asin Formula I, or R and R′ may be limited according to paragraph (i)above;

(n) X₄ may be limited to H, R, OH, OCH₃, —CO₂H and —CO₂R′ with R and R′limited according to paragraph (i) above; and,

(o) X₄ may be limited to H, R, OH, OCH₃, —CO₂H or —CO₂CH₃.

The following specific structures are embodiments of this invention. Insome cases, variability at X₁, X₂, and X₄ is shown with reference tosubstituents identified as R, Z, and Y, which for the purposes of theillustrated compounds are defined below. Although relativestereochemistry is illustrated for each structure, the configuration ofchiral centres may vary according to any of the embodiments based onchirality described above.

Sources of Compounds and Assays for Activity

Pelorol may be obtained from natural sources as taught in the prior artand in the Example 1 herein. Solvent fractionation and/or chromatographymay be employed. It is also possible to modify pelorol or otheravailable compounds such as chrysene derivatives by known chemicalmethodologies to add, remove, or replace substituents in order toproduce components of Formula I. Examples of such derivatization stepsas applied to different compounds of Formula I are shown in more detailbelow.

The presence of SHIP 1 modulating compounds in a preparation may bedetermined by use of a variety of assays, including direct monitoring ofa change in activity of SHIP 1 enzyme such as by the methodologydisclosed in Example 1 and FIG. 1 or by biological assays which may bereadily adapted from known procedures, including cell or animal basedassays which monitor changes in: nitric oxide production from activatedmacrophages; IgE induced mast cell degranulation; LPS induced macrophageactivation; TNF-α expression or activity. In addition, standard assaysfor agents which mediate inflammatory activity in living subjects may beemployed. Adaptation of these assays is facilitated by the availabilityof SHIP 1^(−/−) and SHIP 1^(+/−) mice^(15,16) and bone marrow derivedmacrophages¹⁷. In addition, the availability of anti-SHIP 1 antibodies¹⁸facilitates use of immunoassay formats. Such assays may also be used toassess activity of compounds prepared by total synthesis, as describedherein.

Total Synthesis of Compounds

A synthetic scheme for making pelorol and other compounds of Formula Iis provided herein. Tables (1-2) provide detailed examples of twoembodiments of such a synthesis with examples of different compounds ofFormula I which may be prepared. The compound shown in the Tables thatis identical to pelorol except that the ring adjacent the aromatic ringhas six members, is termed “homopelorol”. Compounds having asix-membered ring are termed “homopelorol analogs”. Compounds having afive-membered ring other than pelorol are termed herein, “pelorolanalogs”.

In the synthesis methods shown in Tables 1 and 2, compounds of FormulaIIA shown therein are conveniently based on sclareolide as a startingmaterial. Appropriate derivatives of sclareolide providing desired R₁-R₄substituents may be employed. In the aromatic compound of Formula IIIshown in the Tables, Nu is preferably lithium. X₂ in the startingcompound of Formula III is preferably an activating group such as —OMeor —NHAc. X₁-X₄ may remain as found in the starting material or beappropriately altered to provide the desired substituents for the endproduct. Protecting groups may be employed on R₁-R₄ or X₁, X₃, or X₄. Anexample of derivatization of the ring comprising L′ in Formula IA toproduce a desired component Q of Formula I is illustrated in Table 2where oxidation (e.g. by treatment with OsO₄ followed by treatment withan acid such as HCl) is performed to provide a ketone substituent on thering. TABLE 1 Synthesis of Pelorol and Pelorol Analogs

TABLE 2 Synthesis of Homopelorol and Homopelorol Analogs

Pharmaceutical Compositions, Dosages, Administration and Indications

Compounds for use in this invention may be formulated intopharmaceutical compositions in any number of ways, which would be knownto a person of skill in the art, all of which are within the scope ofthe invention. The person of skill in the art may be expected to selectappropriate pharmaceutically acceptable salts as well as appropriatepharmaceutically acceptable excipients, diluents, and carriers.

Compounds according to the invention can be provided alone or incombination with other agents (for example, small molecules, peptides,or peptide analogues) in therapeutically- or prophylactically-acceptableamounts, in any pharmaceutically acceptable carrier. Methods well knownin the art for making such pharmaceutical formulations are found in, forexample, “Remington's Pharmaceutical Sciences” (19^(th) edition), ed. A.Gennaro, 1995, Mack Publishing Company, Easton, Pa., incorporated byreference herein. Pharmaceutical formulations according to the presentinvention may, for example, contain excipients, sterile water, orsaline, ethanol, methanol, dimethyl sulfoxide, polyalkylene glycols suchas polyethylene glycol, propylene glycol, or other synthetic solvents,oils of vegetable origin, or hydrogenated napthalenes.

Compounds according to the invention may include hydrophobic compounds,for example, compounds that are substantially insoluble in water, butare freely soluble in solvents such as, for example, ethanol, methanol,dimethyl sulfoxide, or chloroform, or combinations thereof. Formulationscontaining such hydrophobic compounds may be provided using, forexample, micelles, which are formed by amphiphilic compounds undercertain conditions. In aqueous solutions, micelles are capable ofincorporating hydrophobic compounds in their hydrocarbon cores, orwithin the micelle walls. Hydrophobic compounds may also be provided bysolubilization in triglycerides (oils), for example, a digestiblevegetable oil. The solubilized hydrophobic compound in the oil phase maybe dispersed in an aqueous solution and stabilized using emulsifyingagents, if desired. Alternatively, the hydrophobic compound may beprovided in oil and delivered, for example, to the gastrointestinalsystem where bile salts may function as in vivo emulsifiers. Hydrophobiccompounds may also be provided as microemulsions which, like emulsions,are liquid dispersions of oil and water, but have smaller particles withan oil phase in a micelle-like “core.” Hydrophobic compounds accordingto the invention may also be provided together with a polymeric carrier,for example, a carbohydrate such as starch, cellulose, dextran,cyclodextrin, methylcellulose, or hyaluronic acid, or a polypeptide,such as albumin, collagen, or gelatin. Other modes of formulation ofhydrophobic compounds may include liposomes, natural and syntheticphospholipids, or solvents, for example, dimethyl sulfoxide or alcohols.

The pharmaceutical compositions of the invention may be formulated so asto provide controlled release of the active compound(s) over a period oftime. Thus, the formulations could contain, for example, an amount ofthe compound that would be toxic if administered as a single dose, butwhose controlled release does not exceed toxic levels. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers, for example, may be used tocontrol the release of the compounds. Other potentially useful deliverysystems for modulatory compounds according to the present inventioninclude ethylene-vinyl acetate copolymer particles, osmotic pumps,implantable infusion systems, and liposomes.

A “therapeutically effective amount” of a compound is an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result using a compound according to the invention.A therapeutically effective amount is also one in which any toxic ordetrimental effects of the compound are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” of a compound refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired prophylactic result.Typically, a prophylactic dose is used in subjects prior to or at anearlier stage of disease, so that a prophylactically effective amountmay be less than a therapeutically effective amount. Amounts consideredsufficient will vary according to the specific compound used, the modeof administration, the stage and severity of the disease, the age, sex,weight, and health of the individual being treated, and concurrenttreatments.

A preferred range for therapeutically or prophylactically effectiveamounts of the compounds of the invention may be 0.1 nM-0.1M, 0.1nM-0.05M, 0.05 nM-15 μM or 0.01 nM-10 μM. It is to be noted that dosagevalues may vary with the severity of the condition to be alleviated. Forany particular subject, specific dosage regimens may be adjusted overtime according to the individual need and the professional judgement ofthe person administering or supervising the administration of thecompositions. Dosage ranges set forth herein are exemplary only and donot limit the dosage ranges that may be selected by medicalpractitioners. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation.

In general, compounds of the invention should be used without causingsubstantial toxicity. Toxicity of the compounds of the invention can bedetermined using standard techniques, for example, by testing in cellcultures or experimental animals and determining the therapeutic index,i.e., the ratio between the LD50 (the dose lethal to 50% of thepopulation) and the LD100 (the dose lethal to 100% of the population).In some circumstances however, such as in severe disease conditions, itmay be necessary to administer substantial excesses of the compositions.

Conventional pharmaceutical practice may be employed to provide suitableformulations or compositions to administer the compounds to patients,depending on the therapeutic or prophylactic objectives. Any appropriateroute of administration may be employed, for example, systemic,parenteral, intravenous, subcutaneous, transdermal, transmucosal,intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,intracapsular, intraspinal, intracistemal, intraperitoneal, intranasal,aerosol, topical, surgical, or oral administration. The formulationsused may vary according to the chosen route of administration. Thus, fororal administration, the formulations may be in the form of tablets orcapsules; for inhalants, the formulations may be in the form of powders,nasal drops, or aerosols; for transmucosal administration, theformulations may be nasal sprays or suppositories; for transdermaladministration, the formulations may be creams, ointments, salves, orgels; etc.

Therapeutically effective or prophylactically effective amounts of SHIP1 modulators and pharmaceutical compositions of this invention may beadministered to patients in need of treatment or prophylaxis for cancer(neoplastic diseases), other cell proliferative disorders, inflammatorydiseases and immune diseases. Neoplastic diseases include but are notlimited to: leukemias, carcinomas, sarcoma, melanomas, neuroblastoma,capillary leak syndrome and hematological malignancies. Diseases with aninflammatory component include, but are not limited to: rheumatoidarthritis, multiple sclerosis, Guillan-Barre syndrome, Crohn's disease,ulcerative colitis, inflammatory bowel syndrome, psoriasis, graft versushost disease, host versus graft, lupus erythematosis, Alzheimer'sdisease and insulin-dependent diabetes mellitus. Diseases related toinappropriate activation of macrophage-related cells of thereticuloendothelial lineage include osteoporosis.

Pelorol and other compounds having the structure of Formula I exhibitSHIP 1 agonist activity. By activating SHIP 1, such agonists areparticularly useful in the treatment of inflammatory diseases such assepsis/septic shock, colitis, inflammatory bowel syndrome, and thoseinvolving macrophage proliferation or activation; neoplastic diseasessuch as myeloid and lymphoid leukemias; as an immunosuppressive agentsuch as in transplant rejection; hematopoietic disorders; and foraffecting mast cell degeneration such as in the treatment or preventionof allergies.

EXAMPLE 1

In a preliminary screen of 150 marine organism extracts, extracts whichactivated SHIP 1 in an enzyme assay were identified. Assay-guidedfractionation of one of these extracts resulted in the identification ofthe active compound as being pelorol (FIG. 1). The origin and processingof the extracts which tested positive in the screen and the nature ofthe assay were as follows.

Specimens of the brownish sheet sponge Dactylospongia elegans (orderDictyoceratida, family Spongiidae) were collected by hand using SCUBA ata depth of 5-10 m from a protected overhang in Rasch Passage on theouter reef of Madang Lagoon, Papua New Guinea, in January 1995. Freshlycollected sponge was frozen on sight and transported to Vancouver,Canada over dry ice. The sponge was identified and for verification, avoucher sample was placed in the Zoological Museum of Amsterdam (ZMAPOR. 15986). The frozen sponge (120 g) was cut into small pieces,immersed in and subsequently extracted repeatedly with MeOH (3×250 mL).The combined methanolic extracts were concentrated in vacuo and thenpartitioned between EtOAc (4×100 mL) and H₂O (300 mL). The combinedEtOAc extract was evaporated to dryness in vacuo to yield 490 mg of abrownish purple oil, found to contain pelorol.

The assay was performed in 96-well microtitre plates. SHIP 1 enzyme wasproduced with a hemagglutinin and a hexahistidine tag, from a mammalianexpression vector. The His tag was employed to enhance purification.SHIP 1 enzyme (10 ng) was incubated with extract or DMSO for 15 minutesat room temperature before addition of 200 Minositol-1,3,4,5-tetrakisphosphate. The reaction was allowed to proceedfor 20 minutes at 37 degrees C. The amount of inorganic phosphatereleased was then assessed by the addition of malachite green reagentfollowed by an absorbance measurement at 650 nm.

EXAMPLE 2

Pelorol was prepared according to the following scheme, under thespecific conditions described below.

To a stirred solution of 1 (1.00 g, 3.99 mmol) in anhydrous Et₂O (30 mL)was added a freshly prepared 1.6M solution of MeLi in Et₂O (3 mL, 4.8mmol) in portions for 10 min at r.t. and stirring was continued foranother 5 min. The mixture was then treated with 10% HCl (2 mL), thentransferred to a funnel and extracted with ethereal repeatedly. Thecombined extracts was washed with NaHCO₃ and H₂O, dried (MgSO₄),filtered and concentrated. The residue was column chromatographed withhexane/Et₂O (6:4) to give 0.74 g (70%) of 2.

To a stirred, cooled (ice bath) solution of (CF₃CO)₂O (9 mL, 63.85 mmol)in CH₂Cl₂ (40 mL) was added 50% aq H₂O₂ (1.8 mL, 31.66 mmol) and themixture was allowed to stand in an ice bath for 10 min. All subsequentoperations were performed at r.t. The solution was treated with solidNaHCO₃ (5.40 g, 64.28 mmol) for 2 min and after stirring the mixture for8 min, a solution of 2 (1.80 g, 6.76 mmol) in CH₂Cl₂ (54 mL) was added.The resulting mixture was stirred for 30 min and then, after addition ofH₂O (10 mL), was treated with solid NaHCO₃ in portions for 45 min untilthe pH reached 7. Finally, the mixture was extracted with Et₂O. Thecombined extracts were washed with NaHCO₃, H₂O and dried (MgSO₄),filtered and concentrated to give pure 3.

Compound 3 (1 g, 3.6 mmol) was dissolved in 10% solution of KOH in MeOH(1 mL, 1.78 mmol) at 0° C. The resulting mixture was stirred for 10 min.After addition of H2O, the solution was extrated with Et₂O. The extratedwas washed with H₂O, dried (MgSO₄), filtered and concentrated to give0.8 g of 4.

In an oven dried, N₂ flushed 100 mL round bottom flask equipped with amagnetic stirring bar was placed 3.24 g (15 mmol) of PCC, 30 mL ofCH₂Cl₂ and 2.4 g (10 mmol) of 4. The mixture was well stirred at r.t.for 2 hrs and was quenched by adding 30 mL of Et₂O. The resultingsolution was filtered through a thick pad of silica gel and concentratedto give a residue. The residue was column chromatographed with hexane/EA(8:2) to give 1.6 g (67%) of 5.

Sodium hydride (24.6 mg, 0.82 mmol, 80% oil dispersion) and dry THF (5mL) were added to a dry flask equipped with a condenser and dry N₂ flow.To this suspension was added methyl triphenyl phosphonium bromide (0.146g, 0.41 mmol) and the mixture was stirred for 10 min. Then 6 (100 mg,0.41 mmol) in THF (2 mL) was added and the mixture was gently reflux for2 h. The reaction was quenched by adding 2 mL of methanol and thenextracted with Et₂O. After usual work up treatment. 94.3 mg of 7 wasafforded.

A 1.6M solution of tBuLi in pentane (1.74 mL, 2.79 mmol) was addedslowly to a stirred solution of 7 (612.6 mg, 2.52 mmol) in dry THF (20mL) at −78° C. After stirring for 30 min, a solution of 5 (300 mg, 1.26mmol) in dry THF (5 mL) was added. The mixture was further stirred at−78° C. for 2 hrs. Then H₂O (10 mL) was added and the mixture wasextracted with Et₂O (120 mL twice). The combined Et₂O extracts werewashed with sat.brine, dried (MgSO₄) and concentrated to give a residue,which was chromatographed on NP Sepak™ to give 280 mg (55%) of 9.

A solution of 9 (40 mg, 0.1 mmol) in EA (5 mL) was hydrogenated over 10%Pd/C (50 mg) under an atmosphere of hydrogen at r.t. over night.Filtration and concentration gave 37 mg (96%) of 10.

To a stirred solution of 10 (38.8 mg, 0.1 mmol) in CH₂Cl₂ (10 mL), SnCl₄(0.1 mL) was added slowly at −20° C. under argon for 2 min. Theresulting mixture was further stirred for 20 min and then diluted withCH₂Cl₂ (20 mL) and poured into ice. The aqueous phase was extracted withCH₂Cl₂ twice (20 mL) and combined the extracts, washed with saturatedNaHCO₃, saturated brine and dried over MgSO₄. Evaporation to afford 11(28 mg, 76%).

PCC (41.6 mg, 0.192 mmol) was added to 11 (7.4 mg, 0.02 mmol) dissolvedin 2 mL of CH₂Cl₂. The mixture was stirred at gentle reflux for 24 hrsunder Argon. The reaction was diluted with Et₂O (20 mL) and theresulting dark solution was filter through a NP Sepak™. Concentration ofthe filtrates and further purification afford 1.5 mg (20%) of 12.

1.5 mg of 12 was dissolved and stirred in 2 mL of NaOH (10%) solution(containing 0.5 mL THF). 5 mg Iodine is added subsequently and themixture was further stirred for 20 min and acidified by adding 3 mL of10% H₂SO₄. The solution was extracted with 50 mL of Et₂O, washed withsaturated brine and concentrated to afford a residue 13.

38.6 mg (0.1 mmol) of 13 was stirred in CH₂Cl₂ (1 mL) under Argon. BBr₃in CH₂Cl₂ (2.0 mL 1M) was added, and stirring was continued for 1.5 h.The mixture was then poured into H₂O and extracted with CH₂Cl₂ (50 mL).The combined extracts were then dried over MgSO₄, filtered andconcentrated. The residue was purified by NP Sepak™ (hexane:EA=7:3) toafford 14 (25 mg, 70%).

35.8 mg (0.1 mmol) of 14 was dissolved in MeOH (2 mL) containing 5%H₂SO₄. Stirring was continued for 2 hr and the mixture was extractedwith Et₂O, dried over MgSO₄ and concentrated to afford 15.

EXAMPLE 3

The pelorol analog PNSR-15A was synthesized following the methodologiesdescribed in Example 3 using the following scheme.

EXAMPLE 4

The homopelorol analog PNSR-4A was synthesized by the methodologiesdescribed above and according to the following scheme.

EXAMPLE 5

The homopelorol analog PNSR-14A was synthesized by the methodologiesdescribed above and according to the following scheme.

EXAMPLE 6

Homopelorol may be synthesized according to the following scheme basedon the preceding examples and following the methodologies describedabove.

EXAMPLE 7

In addition to causing an increase in activity in the SHIP 1 enzymeassay described for FIG. 1, agonist compounds of Formula I exhibitanti-inflammatory actions on macrophages and mast cells in intactcell-based assays, inhibit nitric oxide production from endotoxinactivated wild-type macrophages and exhibit anti-inflammatory actions onlive subjects. Results obtained for pelorol in NO release and mast cellactivation assays are shown in FIGS. 2 and 3, respectively. Inhibitionof NO release was not observed in SHIP 1−/− macrophages. Pelorolsignificantly reduced IgE induced mast cell degranulation.

Procedures used in the cell and animal based assays are described below.Results for pelorol and various analogs within Formula I are shown inTable 3, including results using the enzyme assay described in Example1.

For the NO release assay, wild-type or SHIP 1−/− macrophage cells werealiquoted into microtitre plates (5×10⁴/well) and activated with 1 g/mLendotoxin (LPS) in the presence or absence of test compound or DMSOcarrier. The cells were incubated at 37° C., 5% CO₂ for 24 hours and theculture supernatant was removed for NO determination using the Griessreagent. Alternatively, J774.1a macrophage cells were treated with 10μg/ml of test compound dissolved in DMSO for 40 minutes prior to theaddition of LPS. Culture supernatants were collected after 24 hr. fordetermination of NO concentration using the Griess reagent.

For the mast cell activation assay, bone marrow derived mast cells wereincubated at 4° C. with anti-DNP IgE for 1 hr. They were then washedtwice with 23° C. Tyrode's buffer, and incubated in the presence of testcompound or vehicle control for 30 minutes before a 15 minute treatmentwith DNP-human serum albumin. The degree of degranulation was determinedby measuring the release of β-hexosaminidase.

For the macrophage TNF-α production assay, J774.1a macrophage cells weretreated with 10 μg/mL of test compound dissolved in cyclodextrin for 40minutes prior to the addition of 100 ng/mL LPS. Culture supernatantswere collected after 2 hr and 5 hr for TNF-α determination by ELISA.

The mouse ear edema (Evans Blue) assay is a standard model for allergicinflammation. Mice were passively sensitized by intravenous injection ofmonoclonal anti-DNP IgE antibody. 24 hours later, 10 μg test compound(right ears) in 20 μl DMSO:Methanol (1:3) or vehicle alone (left ears)were applied 20 minutes followed by application of the inducing agent[20 μl of 0.15% DNFB in acetone:olive oil (4:1)]. Mice were theninjected intravenously with 300 μl 1% Evans Blue. Vascular permeabilitywas measured at 1 hr after application of the inducing agent by visualinspection and quantification of Evans Blue extravasation in the ear. Toquantify the Evans Blue content, ears were harvested at 1 hr post DNFBtreatment and Evans Blue was extracted by incubation in formamide at 37°C. for 24 hr and quantified by spectrophotometry at 620 nm. Earspretreated with carrier alone mounted a prompt anaphylactic reaction inresponse to DNFB challenge. In contrast, SHIP 1 agonists showed a clearinhibition of vascular permeabilization as shown by decreased Evans Blueextravasation.

The mouse ear edema (lymphocyte infiltration assay) is a contacthypersensitivity or ear inflammation model and is a standard in vivomodel for human allergy. Contact hypersensitivity consists of an initialsensitizing phase and an elicitation phase. The latter phase occurs whenthe epidermal cells encounter a particular antigen to which they havepreviously been exposed and is characterized by localized immune cellinfiltration, inflammation, and edema. In this assay, female 4 week old(20 g) Balb/c mice were sensitized to the haptenizing agent,2,4-dinitrofluorobenzene (DNFB) by shaving their abdominal region withan electric razor before applying 25 μl of 0.5% DNFB in acetone:oliveoil (4:1, v/v) to the abdominal wall for two consecutive days. Four daysafter the second application, mice were lightly anesthesized withhalothane before being challenged (treated) epicutaneously on each sideof the right and left ear with 10 μl of 0.2% DNFB. All mice received a500 μl intraperitoneal (i.p.) injection of [³H]-methyl thymidine insterile saline (1 μCi/g body weight) 24 hours before epicutaneouschallenge with DNFB. Thirty minutes prior to DNFB challenge, the rightand left ears were pretreated with test compound in DMSO:methanol (1:3,v/v) or vehicle alone, respectively. Twelve hours following DNFBchallenge, mice were sacrificed by CO₂ asphyxiation and 8 mm diameterplugs were taken from each ear and digested in 500 μl Solvable™ at 60°C. for 10-12 hours. Samples were decolourized by the addition of H₂O₂and analyzed for radiolabelled leukocyte infiltrates by standard liquidscintillation counting.

The colitis assay is based on determining whether a test compoundprotects mice from TNBS (trinitrobenzene sulfonic acid) inducedinflammation. Test compound (10 mg/kg) or vehicle control was injectedintraperitoneally into mice just prior to a TNBS enema administration.After 2 days, the colons of the vehicle treated mouse were severelyinflamed while the SHIP 1 agonist treated mouse had no signs ofinflammation. TABLE 3 SHIP enzyme Macrophage NO Macrophage TNFα Mastcell Mouse ear edema Mouse ear edema assay production productionactivation (evans blue assay) (leukocyte infiltration assay) colitisPelorol +++ +++ +++ +++ +++ ND +++ Dimethoxypelorol + +++ ND ND ND ND NDPNSR-4A insol ND ND ND +++ ND ND PNSR-15A ND ND ND ND ND ++ ND PNSR-16AND ND +++ ND ND ++ ND PNSR-17A ND ND ++ ND ND ND ND PNSR-18A ND ND +++ND ND ND ND

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of skill in the artin light of the teachings of this invention that changes andmodification may be made thereto without departing from the spirit orscope of the appended claims. All patents, patent applications andpublications referred to herein are hereby incorporated by reference.

REFERENCES

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1. A compound of Formula I or a salt thereof,

wherein; R₁ and R₂ are independently selected from the group consistingof: —CH₃, —CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′; R₃ and R₄are independently selected from the group consisting of: H, —CH₃,—CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′; Q is a —CH₂—; X₁, X₂,X₃, and X₄ are independently selected from the group consisting of: H,R, OH, —OR, —CO₂H, —CO₂R′, F, Br, Cl, I, —CN, —SO₃H, —OSO₃H, NO₂, NH₂,—NHR, and —NR₂; where R is a linear, branched, or cyclic, saturated orunsaturated one to ten carbon alkyl group that is unsubstituted or issubstituted with one or more of: OH, ═O, SH, F, Br, Cl, I, NH₂, —NHR′,—NR′₂, NO₂, —CO₂H, —CO₂R′, and epoxide; and R′ is a linear, branched, orcyclic, saturated or unsaturated one to ten carbon alkyl group that isunsubstituted or substituted with one or more of: OH, ═O, SH, F, Br, Cl,I, NH₂, —NHR″, —NR″₂, NO₂ and —CO₂H where R″ is a linear, branched, orcyclic, saturated or unsaturated one to ten carbon alkyl group;providing that the compound does not have the precise structure ofpelorol 2-5. (canceled)
 6. The compound of claim 1, wherein R₁ ismethyl, ethyl, —CH₂OH, or —CH₂OR′.
 7. The compound of claim 1, whereinR₂ is methyl, ethyl, —CH₂OH, or —CH₂OR′.
 8. The compound of claim 1,wherein R′ in R₁ is limited to methyl, ethyl, propyl or butyl.
 9. Thecompound of claim 1, wherein R′ in R₂ is limited to methyl, ethyl,propyl or butyl.
 10. The compound of claim 1, wherein R₁, R₂, R₃ and R₄are methyl.
 11. The compound of claim 1, wherein X₁ is H, OH, R, OR,—CONH₂, —CONHR′, or —COR′.
 12. The compound of claim 1, wherein X₂ is H,OH, R, OR, —CONH₂, —CONHR′, or —COR′.
 13. The compound of claim 1,wherein X₃ is H, OH, R, OR, —CONH₂, —CONHR′, or —COR′.
 14. The compoundof claim 1, wherein R and R′ in one or more of X₁, X₂, and X₃ arelimited to methyl, ethyl, propyl and butyl.
 15. The compound of claim 1,wherein X₁ is H, OH, or —OCH₃.
 16. The compound of claim 15, wherein X₂is H, OH, or OCH₃.
 17. The compound of claim 10, wherein X₁ and X₂ areindependently selected from H and OH.
 18. The compound of claim 1,wherein X₃ is H, OH, or OCH₃.
 19. The compound of claim 1, wherein X₄ isH, R, OH, OR, CO₂H or CO₂R′.
 20. The compound of claim 17, wherein X₃and X₄ are independently selected from H, methyl, ethyl, propyl andbutyl.
 21. The compound of claim 17, wherein X₃ is H and X₄ is methyl.22. The compound of claim 1, selected from: PNSR-16A and PNSR-18A. 23.The compound of claim 1, having the configuration S, R, R, S at C-5,C-8, C-9 and C-10 respectively.
 24. The compound of claim 1, having theconfiguration R, S, S, R at C-5, C-8, C-9 and C-10 respectively. 25-33.(canceled)
 34. A pharmaceutical composition comprising apharmaceutically acceptable carrier and one or more compounds of FormulaI or pharmaceutically acceptable salts thereof,

wherein; R₁ and R₂ are independently selected from the group consistingof: —CH₃, —CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′; R₃ and R₄are independently selected from the group consisting of: H, —CH₃,—CH₂CH₃, —CH₂OH, —CH₂OR′, —CHO, —CO₂H, and —CO₂R′; Q is a —CH₂—; X₁, X₂,X₃, and X₄ are independently selected from the group consisting of: H,R, OH, —OR, —CO₂H, —CO₂R′, F, Br, Cl, I, —CN, —SO₃H, —OSO₃H, NO₂, NH₂,—NHR, and —NR₂; where R is a linear, branched, or cyclic, saturated orunsaturated one to ten carbon alkyl group that is unsubstituted or issubstituted with one or more of: OH, ═O, SH, F, Br, Cl, I, NH₂, —NHR′,—NR′₂, NO₂, —CO₂H, —CO₂R′, and epoxide; and R′ is a linear, branched, orcyclic, saturated or unsaturated one to ten carbon alkyl group that isunsubstituted or substituted with one or more of: OH, ═O, SH, F, Br, Cl,I, NH₂, —NHR″, —NR″₂, NO₂ and —CO₂H where R″ is a linear, branched, orcyclic, saturated or unsaturated one to ten carbon alkyl group.
 35. Thepharmaceutical composition of claim 34, wherein R₁, R₂, R₃ and R₄ aremethyl.
 36. The pharmaceutical composition of claim 35, wherein X₁ andX₂ are independently selected from H and OH.
 37. The pharmaceuticalcomposition of claim 36, wherein X₃ is hydrogen and X₄ is methyl. 38.(canceled)